| 闫凡,黄彩虹.下调微小 RNA-183表达对肾母细胞瘤 SK-NEP-1细胞增殖凋亡的影响及机制[J].安徽医药,2022,26(3):553-557. |
| 下调微小 RNA-183表达对肾母细胞瘤 SK-NEP-1细胞增殖凋亡的影响及机制 |
| Effect of down-regulation of miR-183 expression on proliferation and apoptosis of nephro? blastoma SK-NEP-1 cells and its mechanism |
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| DOI：10.3969/j.issn.1009-6469.2022.03.031 |
| 中文关键词: 肾母细胞瘤 微小 RNA-183 凋亡 胱天蛋白酶 -3 PDCD4 |
| 英文关键词: Wilms Tumor Mir-183 Apoptosis Caspase-3 PDCD4 |
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| 中文摘要: |
| 目的探讨下调微小 RNA(miR)-183表达对肾母细胞瘤 SK-NEP-1细胞增殖凋亡的影响及机制。方法 2018年 12月至 2019年 7月，体外培养 SK-NEP-1细胞，分为对照组(细胞正常培养)、抑制剂阴性对照组( Anti-NC组)(转染 Anti-NC至 SKNEP-1细胞)、 miR-183抑制剂组( Anti-miR-183组)(转染 Anti-miR-183至 SK-NEP-1细胞)、 Anti-miR-183+小干扰 RNA阴性序列(si-NC)组(共转染 Anti-miR-183和 si-NC至 SK-NEP-1细胞)和 Anti-miR-183+程序性细胞死亡 4小干扰 RNA(si-PDCD4)组(共转染 Anti-miR-183和 si-PDCD4至 SK-NEP-1细胞)，实时荧光定量逆转录聚合酶链反应( qRT-PCR)测定 miR-183，MTT法检测细胞增殖变化，平板克隆实验检测细胞克隆能力，流式细胞术测定细胞凋亡变化，蛋白质印迹法( Western blotting)检测细胞中活化胱天蛋白酶 -3(cleaved-caspase-3)和 PDCD4蛋白水平。双荧光素酶报告基因实验验证 miR-183与 PDCD4的靶向关系。结 |
| 英文摘要: |
| Objective To investigate the effect of down-regulation of miR-183 on proliferation and apoptosis of nephroblastoma SKNEP-1 cells and its mechanism.Methods From December 2018 to July 2019, SK-NEP-1 cells were cultured in vitro and assigned into control group (SK-NEP-1 cells were cultured normally), inhibitor negative control (Anti-NC) group (SK-NEP-1 cells were transfected with Anti-NC), miR-183 inhibitor (Anti-miR-183) group (SK-NEP-1 cells were transfected with Anti-miR-183), Anti-miR-183+small interfering RNA negative sequence (si-NC) group (SK-NEP-1 cells were co-transfected with Anti-miR-183 and si-NC) and Anti-miR-183+ programmed cell death 4 small interfering RNA (si-PDCD4) group (SK-NEP-1 cells were co-transfected with Anti-miR-183 and siPDCD4). Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect miR-183. Tetrathiazolyl blue (MTT) assay was used to detect cell proliferation. Plate cloning assay was used to detect cell cloning ability. Apoptotic changes were measured by flow cytometry. Western blotting was used to detect the protein levels of cleaved-caspase-3 and PDCD4. The dual luciferase reporter gene experiment verified the targeting relationship between miR-183 and PDCD4. Results The expression level of miR-183 in the Anti-miR-183 group was (0.32±0.04), which was significantly lower than that in the Anti-NC group (0.98± 0.11) (P<0.05). The cell proliferation activity of the Anti-miR-183 group was (0.20±0.02) and the number of clones was (213.69± 20.94), which were lower than those of the Anti-NC group [(0.40±0.03), (312.87±22.28)] (P<0.05). The apoptotic rate of the Anti-miR183 group was (16.47±1.58)% and the level of cleaved-caspase-3 protein was (0.69±0.09), which were higher than those of the Anti-NC group [(2.36±0.32)%, (0.27±0.04), respectively] (P<0.05). There was no statistically significant difference in the detection indicators between the control group and the Anti-NC group (P>0.05). PDCD4 was identified as the target gene of miR-183, and the level of DCD4 protein in the Anti-miR-183 group was (0.89±0.07), which was significantly higher than that of the Anti-NC group (0.37±0.06) (P< 0.05). The cell proliferation activity of Anti-miR-183+si-PDCD4 group was (0.32±0.04), the number of clones was (286.47±20.65), andthe level of PDCD4 protein was (0.45±0.05), all higher than those in the Anti-miR-183+si-NC group [(0.21±0.03), (216.20±13.68), (0.86±0.09), respectively] (P<0.05); the apoptosis rate of Anti-miR-183 group was (10.79±1.28)%, and the level of cleaved-caspase-3 protein was (0.30±0.03), which were both lower than those in the Anti-miR-183+si-NC group [(17.93±1.64)%, (0.72±0.08), respectively] (P<0.05).Conclusion Downregulation of miR-183 inhibits proliferation and induces apoptosis of nephroblastoma SK-NEP-1 cells by targeting PDCD4. |
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